CN103201057A

CN103201057A - Blue-colored gold nanoparticles for immunological measurement, process for production of same, and measurement method using same

Info

Publication number: CN103201057A
Application number: CN2011800533886A
Authority: CN
Inventors: 加藤佑弥; 伊藤大辅; 木谷佳子
Original assignee: Tanaka Kikinzoku Kogyo KK
Current assignee: Tanaka Kikinzoku Kogyo KK
Priority date: 2010-11-05
Filing date: 2011-11-04
Publication date: 2013-07-10
Anticipated expiration: 2031-11-04
Also published as: EP2636469A4; AU2016216707B2; CA2816674A1; WO2012060456A1; JP2012112042A; KR101729810B1; JP5358648B2; AU2011324320A1; EP2636469A1; IL226071A0; KR20130142145A; CN103201057B; US20130224885A1; AU2016216707A1; US9360431B2; IL226071A; AU2011324320B2; CA2816674C

Abstract

A nuclei formation step of reacting an organic buffering agent having a piperazine ring with a solution of a first gold salt to form nuclear gold nanoparticles and a growth step of growing the nuclear gold nanoparticles by simultaneously adding a solution of a second gold salt and an organic acid having reducing properties to the solution of the nuclear gold nanoparticles to cause the reaction of these components are carried out. Thus, it becomes possible to readily produce gold nanoparticles which can be dispersed into a colloid solution having a blue color as determined by visual examination and each of which comprises an organic buffering agent having a piperazine ring, gold and an organic acid having reducing properties. The gold nanoparticles thus produced can be used as labeling particles for use in immunological measurement methods.

Description

Immunologic assay is with blue gold nano grain, its manufacture method and the assay method that uses this blueness gold nano grain

Technical field

The present invention relates to the colloidal solution of blue gold nano grain and blue gold nano grain, they all have highly distinct colour rendering, have stable durability and excellent identity simultaneously, and can be used as immunologic assay with labelled reagent or protein staining agent.The invention still further relates to the method for making blue gold nano grain of the present invention, and the detection kit and the assay method that use this blueness gold nano grain.In addition, the present invention relates to the immunologic assay mark substance, in this immunologic assay, blue gold nano grain of the present invention is used as the mark substance in the immunoassay system.

Background technology

In recent years, come simple and easy external diagnosis reagent case or the portable diagnostic device of the antigen in the test sample liquid as the atopy of utilizing antibody to have, immuno-chromatographic test paper strip formula immunologic assay method becomes more important.Particularly, in order to detect the infection of pathogen such as whether having influenza virus or bacterium, the simple and easy multinomial testing tool based on immunochromatographic method is studied and researched and developed.

Colloidal metal particle or latex particle are often used as the insoluble carrier for the immunologic assay method.Wait for mark substance in order to support protein securely, latex particle needs complicated manufacturing steps such as chemical functional group's modification.Therefore, the preferred use colloid gold particle that can easily support material to be marked and can make easily with low cost.

Because antibody is only needed easy operation and utilizes it only to check to need the short time by the inspection medicine of the immunochromatographic method of insoluble carrier indicium, therefore be extensive use of, yet, compare with EIA, because the common sensitivity of this inspection medicine is low, when the result is positive, can observed line be unsharp therefore.

In order to address this problem, developed multiple metallic colloid, these metallic colloids have higher sensitivity than the conventional colloidal metal particle that has dropped into practical application, and are suitable as immunologic assay with labelled reagent or protein staining agent.

Patent documentation 1 provides and supported the average grain diameter that platinum obtains by the surface at colloidal metal particle (average grain diameter is 30-100nm) is the colloidal metal particle of 50-150nm, this is because the little and insufficient colour developing of average grain diameter of colloid paper tinsel particle is not suitable for being used in the immunochromatographic method.This colloidal metal particle is to form colloid gold particle, to reduce (referring to the patent documentation 1) that chloroplatinic acid prepares then under the gained colloid gold particle by reduction gold chloride in solvent.

Patent documentation 2 provides the more highly sensitive colloidal metal particle that has that obtains by above-mentioned colloidal metal particle is improved.That is, provide and supported the colloidal metal particle that average grain diameter is the platinum of 5nm (average grain diameter is 30-100nm) thereon.This colloidal metal particle is that to be manufactured by the following method prepared: the amount of the amount of the reducing agent that adds during with preparation colloidal metal particle in medium and the reducing agent that adds in the reduction of colloidal metal particle and when supporting platinum is adjusted to preset range; wherein, described medium does not comprise protective colloid formation agent basically.The example that this protective colloid forms agent comprises water-soluble high-molecular substance (as PVA, PVP and gelatin etc.), surfactant and high-molecular chelating agent (referring to patent documentation 2).

Another kind of method as the sensitivity that improves immunology and immunocytology deagnostic test, provide a kind of like this method: with alkyl hydrosulfide coating aurosol ultra-fine grain, to give this aurosol surface with the specific hydrophobic hydrophil balance, thereby prevent the aggegation that is caused by salt, this makes the non-specific interaction between described aurosol surface and the exogenous proteins minimize (referring to patent documentation 3).

On the other hand, with in the Extrasomatic diagnostics, higher sensitivity is improved and shown to the red spherical colloid gold grain of having put on market in cyesiognosis.Collaurum need have: the particle diameter that is suitable for desired use; Sharp-pointed particle diameter distributes; And uniform spheroidal, its manufacturing process is just under development.

Patent documentation 4 comprises: add first reducing agent (citrate) to the solution of the first gold medal salt and become the stage with the karyomorphism that forms colloid nuclear particle (average grain diameter is 12-17nm); And add the second gold medal salt and second reducing agent (ascorbate) in the solution of this colloid nuclear particle simultaneously so that the growth phase of colloid nucleus growth.This growth phase carries out once at least.The average grain diameter of described colloid gold particle is more than or equal to 17nm and less than 55nm in primary growth phase; Be more than or equal to 55nm and less than 110nm in secondary growth phase; Be 111-220nm in growth phase for the third time.The standard deviation of described particle diameter 10% with interior (referring to patent documentation 4).

Only checking under the situation of a project such as the pregnancy check kit that be used for to check gestation whether etc., when visual judgements, only need a kind of labelled reagent of use.Recently, in the virus checking such as the infection of flu shape or respiratory tract infection, need identify that should carry out multiple inspection this moment to Causative virus.Therefore, in order to alleviate patient and medical personnel's burden, developed multiple check system.

For example, although known have a kind of lateral flow immunoassays that can detect a plurality of viruses (rotavirus, calicivirus, coronavirus, adenovirus and enterovirus etc.) by using a kind of checking tool, yet this mensuration has following problem: many visual judgements that detection line is easy to lead to errors.

When by the use immunochromatographic method virus in the respiratory tract infection being checked, developed a kind of like this inspection method, this method comprises: with a corpse or other object for laboratory examination and chemical testing treatment fluid nasal mucus, phlegm or nasal cavity swab liquid are carried out preliminary treatment, be suitable for checking the sample for reference of a plurality of respiratory tract infection with preparation; And by (for example using first checking tool, be used for to check the instrument of influenza infection) and second checking tool a plurality of checking tools such as (instruments that for example, is used for inspection adenovirus infection or RS virus infections) each several part of gained sample for reference is analyzed (referring to patent documentation 5).

In addition, developed a kind of assay method that comprises immunochromatographic method, this immunochromatographic method has strong decision-making ability by the particle of labelled antibody that use has random color, and can measure two or more determination objects simultaneously by using the two or more particles of labelled antibody.More specifically, be positioned at about 550nm, redness by using TRITC(to absorb maximum) and FITC(absorption maximum be positioned at about 500nm, orange) etc. fluorescent dye and simultaneously hCG and LH are measured (referring to patent documentation 6).

When causing judging by accident or the possibility of mistaken diagnosis by using a kind of checking tool to carry out simultaneously with visual judgement that multinomial visual inspection is looked into and employed labelled reagent or protein staining agent when being same color or similar color, having.For erroneous judgement or the mistaken diagnosis that prevents visual judgement, need carry out visual judgement by high labelled reagent or the protein staining agent of identity of using color.

When having two kinds of colors, the color of their identity when being used in combination and different.Because red and blue by visual mutual differentiation that can height, therefore, they are used to various differentiation purposes, as distinguishing men and women or differentiation hot water (red) and water (indigo plant).The colloid gold particle of conventional input practical application is red spherical particle.If color blue colloid gold particle different, that namely identity is high for red as labelled reagent or protein staining agent, is inferred that then erroneous judgement or the misjudgement by visual judgement can obviously reduce.Yet blue colloid gold particle does not drop into practical application as yet.

In patent documentation 7-9, the metal nanoparticle that light absorption wavelength character is changed to the size by changing metal nanoparticle, style, configuration/shape etc. is described.

According to patent documentation 7 and 8, blue gold nano grain has the configuration/shape of gold nanoshell, nanometer rods, nanotube or nanoprisms particle; Described gold nano grain is made in the following manner: (1) adds reducing agent to yellow silver nano-grain solution (containing protective agents such as polyvinylpyrrolidone or ethylene glycol), and the gained mixture is refluxed under about 100 ℃; (2) in the reactant mixture of such process backflow, inject golden salting liquid, make its reaction; And (3) make this reactant mixture filter by the millipore filter of 0.2 μ m after being cooled to normal temperature, like this gold nano grain of Huo Deing only its superficial layer formed by gold (gold nanoshell).According to these documents, gold nanorods, gold nanotubes or gold nano prism are when forming gold nano grain, by using cetab (bromide) (C ₆TAB) etc. surfactant obtains.In these documents not to the limited description of granular size.Comprise in these documents described particle as the description of used for cosmetic pigment, but not about with described particle as the labelled reagent in the immunoassays or the description (referring to patent documentation 7 and 8) of protein staining agent.

Patent documentation 9 has been described by containing C1 ₆The surfactant of TAB(ammonium salt) the bar-shaped gold nano grain that uses reducing agent (amine) to reduce gold ion in the aqueous solution and obtain.The described amine that the aspect ratio of described gold nano grain (major axis/minor axis) can be used in combination by adjusting and the mixed proportion of described ammonium salt are controlled.By such mode, obtain aspect ratio and be 2 to 11 and the absorbing wavelength peak area be the gold nanorods of 658nm to 1200nm.According to this description, these gold nanorods can be used as inspection medicine (referring to patent documentation 9).

Because so the gold nano grain that obtains contains C ₁₆TAB is as surfactant, and therefore, they are not suitable for directly supporting (modification) and detect protein such as antibody.Because this gold nano grain need be removed or complex operations such as permutation table surface-active agent, therefore, as the mark substance that in the immunologic assay method, is used as the protein that checks medicine, preferred this gold nano grain.In addition, owing to have C ₆Therefore TAB toxicity considered from the angle of operation, not preferred this gold nano grain.

Non-patent literature 1 has been described the colloid that is rendered as glaucous rod shape gold nano-crystal.This rod shape gold nano-crystal has complicated three-dimensional structure, has 1-8 projection, and the crystalline size that comprises projection is that the length of 30nm to 50nm(projection is that about 15nm to 25nm, width are about 8nm).This three-dimensional dendritic gold nano-crystal is by aqueous solution of chloraurate and be that the organic acid (HEPES, HEPPSO or PIPES etc.) of Good ' s buffer composition at room temperature reacts and obtains (non-patent literature 1) with high yield (92%).

Yet its crystal size of the colloid that is rendered as glaucous dendritic nanocrystal that obtains in non-patent literature 1 is 30nm to 50nm, and this is not required size.Therefore, even used as the immunochromatography diagnosticum, inadequate colour developing also can hinder visual judgement smoothly.

From the correlation technique document, can clearly learn, be rendered as the colloid of glaucous gold nano-crystal because its colloidal particle size less relatively (for about 30nm to 50nm), so and be not suitable for use in the labeled vector of immunochromatography diagnosticum.In addition, be called as multiway shape (multipod-shaped), colloid dendritic or golden flat sugared shape uses the dimensionally stable agent mostly, these dimensionally stable agent are difficult to utilize protein to finish the direct modification of gold nano grain.

The prior art document

Patent documentation

Patent documentation 1:JP-A-2003-262638

Patent documentation 2:JP-A-2005-233744

Patent documentation 3:JP-A-6-116602

Patent documentation 4:JP-A-2007-321232

Patent documentation 5:JP-A-2008-164403

Patent documentation 6:JP-A-10-132817

Patent documentation 7:JP-A-2008-545884

Patent documentation 8:JP-A-2009-501786

Patent documentation 9:JP-A-2006-118036

Non-patent literature

Non-patent literature 1:Chem.Mater.2007,19,2823-2830

Non-patent literature 2:Langmuir2005,21,2012-2016

Non-patent literature 3:J.Phys.Chem.B2006,110,19291-19294

Non-patent literature 4:Nano Lett.2006,6,683-688

Summary of the invention

The problem that the present invention need solve

The purpose of this invention is to provide blue gold nano grain, colloidal solution and following so blue gold nano grain by this gold nano grain being scattered in the blue gold nano grain that obtains in the medium, this blueness gold nano grain shows highly distinct blueness when visual, excellent quality stability, storage stability and identity, can be used as labelled reagent or the protein staining agent of immunologic assay, and because the easy identification with the red different of routine; And the objective of the invention is to solve the manufacture method of relevant blue gold nano grain, the relevant problem of assay method of measuring the inspection kit of accuracy raising and using this inspection kit by using this blueness gold nano grain to make.

More than be not suitable for use in the carrier of immunochromatography diagnosticum because of following two problems with the blue gold nano grain in the non-patent literature for referencial use 1 to 3:

1. the size of these blue gold nano grains is not suitable for immunologic assay.With regard to average grain diameter, the particle diameter that is suitable for immunochromatographic method reagent is about 40nm to 100nm.According to non-patent literature 1, described particle diameter is about 30nm.

2. they contain the dimensionally stable agent.Contain the dimensionally stable agent at the three-dimensional rod shape gold nano grain described in the non-patent literature 2 to 4, to control their shape.This dimensionally stable agent has prevented the direct modification of protein to gold nano grain.

Be used to multinomial when measuring simultaneously when implementing multiple inspection and conventional red gold nano grain and painted latex particle, because the particle diameter of gold nano grain and latex particle different (size of the latex particle that adopts is greater than gold nano grain usually), therefore in immunochromatographic method, be difficult to select the aperture to be fit to the immunochromatography carrier of these two kinds of particles.Therefore, need to use two kinds of different colloid gold particles of color material that serves as a mark, these two kinds of colloid gold particles can easily support protein etc. and be labeled material, and not expensive.

In order to address the above problem, the size of the present inventor by increasing particle is so that it is suitable for immunologic assay and the dimensionally stable agent by selecting to allow with protein gold nano grain directly to be modified, thereby successfully provide the blue gold nano grain of the carrier that is suitable for the immunochromatography diagnosticum, the blue nanogold particle of the multinomial detection reagent in the multinomial detection that can be used for being undertaken by immunochromatographic method more specifically, is provided.

The method of dealing with problems

The invention provides the blue gold nano grain that is suitable for immunologic assay, it makes protein become easily to the modification of this gold nano grain, is suitable as multinomial detection reagent simultaneously most.

Particularly, blue gold nano grain of the present invention is by being the organic acid that contains piperazine ring of Good ' s buffer solution component (as HEPES etc.), Au(gold) and the organic acid (as ascorbic acid and citric acid etc.) with reproducibility constitute, this gold nano grain is rendered as blueness when visual, and has the flat sugared shape of gold.

Consider from the angle of the stability and durability of in bright gay color property, stability and durability and colloid, the average grain diameter of blue gold nano grain of the present invention is 20nm to 200nm, be preferably 40nm to 180nm, the various practical standpoints of the remarkable identity when checking from comprising are considered, most preferably are 50nm to 120nm usually.Optimum scope is 60nm to 100nm.Be separated in the liquid of colloid at blue gold nano grain, described blue gold nano grain is blue feature when having visual observation.

Term " average grain diameter " expression that the present invention uses is definite value by the nuclear jut that comprises the blue gold nano grain that the back will describe.In blue gold nano grain of the present invention, the length of nuclear jut is preferably 5nm to 50nm.The number of projection is that every nuclear is more than 4.

In containing the colloid aqueous solution of with good grounds blue gold nano grain of the present invention, the average grain diameter of colloid gold particle is 20nm to 200nm, is preferably 40nm to 180nm, most preferably is 50nm to 120nm usually, and optimal is 60nm to 100nm.Average grain nuclear footpath is 20nm to 60nm.The colloid aqueous solution that contains with good grounds blue gold nano grain of the present invention is characterised in that: in ultraviolet-visible absorption spectroscopy, it has maximum absorption wavelength in the scope of 570nm to 750nm.By with gold nano grain contained in the collaurum aqueous solution of the present invention as the mark substance in the immunochromatographic method, then available height distinguishes over red blueness and detects.This makes and can implement immunochromatography under the situation that reduces the mistaken diagnosis case and learn detection in multinomial detection simultaneously.It should be noted that the dispersion of subparticle (particularly gold nano grain) in the water equal solvent of statement " according to the colloid aqueous solution of blue gold nano grain of the present invention " expression nanoscale (nm).In brief, the blue gold nano grain of the flat sugared shape of gold that the present invention successfully provides colloidal solution and the manufacture method thereof of blue nano particle, blue gold nano grain and has been suitable for immunologic assay, it makes protein become easily to the modification of blue gold nano grain, and it is suitable as multinomial detection reagent most simultaneously.

The invention provides blue gold nano grain, its manufacture method with and using method.Gold nano grain of the present invention has following feature:

(a) of the present invention first be characterized as blue gold nano grain, comprise that average grain diameter is the gold nano grain of 20nm to 200nm;

(b) of the present invention second be characterized as according to (a) described blue gold nano grain, wherein, the maximum wavelength of its ultraviolet-visible absorption spectroscopy is in the scope of 570nm to 800nm;

(c) the of the present invention the 3rd be characterized as according to (a) or blue gold nano grain (b), wherein, described gold nano grain is the flat sugared shape particle of grafting shape particle, multiway shape particle or gold with three-dimensional protrusion;

(d) the of the present invention the 4th be characterized as according to any described blue gold nano grain in (a) to (c), it obtains by the periphery growth that makes the nuclear that is made of gold nano grain; And

(e) the of the present invention the 5th be characterized as according to any described blue gold nano grain in (a) to (d), its average grain nuclear is of a size of 20nm to 60nm, average grain diameter is that projection and the projection length that 50nm to 120nm, each nuclear have more than 4 is 5nm to 50nm.

Gold nano grain of the present invention is scattered in the medium such as water and the colloid that forms has following feature:

(f) the of the present invention the 6th colloidal solution that is characterized as blue gold nano grain, it comprises: the blue gold nano grain described in the claim 1; The organic acid that contains piperazine ring for Good ' s buffer solution component; And the organic acid with reproducibility, and it is separated into colloidal solution.

Particularly the manufacture method of gold nano grain of the present invention has following feature:

(g) the of the present invention the 7th be characterized as the method for making blue gold nano grain, comprising: karyomorphism becomes step, wherein, examines gold nano grain with the solution reaction of the first gold medal salt to form by making to the organic acid that contains piperazine ring of Good ' s buffer solution component; Growth step, wherein, the solution by in the solution of this nuclear gold nano grain, adding the second gold medal salt simultaneously and have the organic acid of reproducibility and make its reaction, thus this nuclear gold nano grain is grown;

(h) the of the present invention the 8th method that is characterized as according to the blue gold nano grain of (g) described manufacturing, wherein, described growth step be more than or equal to 10 ℃ and less than 40 ℃ reaction temperature under carry out;

(i) the of the present invention the 9th be characterized as according to (g) or (h) method of the blue gold nano grain of described manufacturing, wherein, the described organic acid concentration in the described growth step is 0.075mM to 0.15mM;

(j) the of the present invention the tenth method that is characterized as according to the blue gold nano grain of (i) described manufacturing, wherein, the described organic acid that contains piperazine ring for Good ' s buffer solution component is for being selected from by 2-[4-(2-ethoxy)-1-piperazinyl] ethyl sulfonic acid, 4-(2-ethoxy)-1-piperazine propane sulfonic acid, 4-(2-ethoxy) piperazine-1-(2-hydroxy propane-3-sulfonic acid), piperazine-1,4-two (2-ethanesulfonic acid), 3-[4-(2-ethoxy)-1-piperazinyl] propane sulfonic acid and piperazine-1, the organic acid of more than one of the group that 4-two (2-hydroxyl-3-N-morpholinopropanesulfonic acid) constitutes;

(k) the of the present invention the 11 method that is characterized as according to the blue gold nano grain of (g) described manufacturing, wherein, described organic acid with reproducibility is more than one the organic acid that is selected from the group that is made of tartaric acid, tartrate, tannic acid, tannate, ascorbic acid, ascorbate, citric acid and citrate; And

(l) the of the present invention the 12 method that is characterized as according to the blue gold nano grain of (g) described manufacturing, wherein, in described growth step, describedly be used in combination for the organic acid that contains piperazine ring and the described organic acid with reproducibility of Good ' s buffer solution component.

Next, particularly the conduct of the present invention mark substance that is used for immunologic assay has following feature:

(m) the of the present invention the 13 mark substance that is characterized as for immunologic assay, it comprises any described blue gold nano grain in (a) to (e);

(n) the of the present invention the 14 be characterized as according to (m) described mark substance for immunologic assay, it comprises at least two kinds of variform gold nano grains;

(o) the of the present invention the 15 be characterized as according to (n) described mark substance for immunologic assay, it comprises at least two kinds of variform gold nano grains, that is: spherical gold nano grain and grafting shape, multiway shape or the flat sugared shape gold nano grain of gold with three-dimensional protrusion; And

(p) the of the present invention the 16 be characterized as a kind of immunologic assay method, it is used as mark substance with any described blue gold nano grain in (a) to (e).

Described problem of the present invention can be resolved by the formation that adopts the invention described above.

The beneficial effect of the invention

Because the average grain diameter of blue gold nano grain of the present invention is 20nm to 200nm, be preferably 40nm to 180nm, most preferably be 50nm to 120nm usually, optimum is 60nm to 100nm, therefore, this blueness gold nano grain can provide the particle diameter that is suitable for the immunochromatography diagnosticum most.

Be used in combination to prepare with spherical red gold nano grain etc. and have the immunochromatography diagnosticum of judging line more than two.Because this can make the visual judgement in the multiple inspection become easy and accurate, has therefore prevented mistaken diagnosis or erroneous judgement.

In addition, blue gold nano grain of the present invention can be easy to by protein modification, so they make it possible under the situation of desensitization not the result be judged accurately.Therefore, described nano particle has excellent performance as the immunochromatography diagnosticum.

In addition, the immunochromatography diagnosticum ratio by blue gold nano grain preparation of the present invention is more cheap by the prepared immunochromatography diagnosticum of the particle that obtains by additive method.

Description of drawings

[Figure 1A] Figure 1A is shape and the rough big or small transmission electron microscope image that an example of blue gold nano grain of the present invention is shown.

[Figure 1B] Figure 1B is shape and the rough big or small transmission electron microscope image that another example of blue gold nano grain of the present invention is shown.

[Fig. 2 A] Fig. 2 A is the transmission electron microscope image that an example of the preceding blue gold nano grain of the present invention of growth is shown.

[Fig. 2 B] Fig. 2 B is the transmission electron microscope image that an example of the blue gold nano grain of the present invention after the growth is shown.

[Fig. 3 A] Fig. 3 A is for illustrating the transmission electron microscope image of another example of the preceding blue gold nano grain of the present invention of growth with 20 times enlargement ratio (length of figure medium scale is 50mm).

[Fig. 3 B] Fig. 3 B is the transmission electron microscope image of blue gold nano grain that Fig. 3 A after the growth is shown with 50 times enlargement ratio (length of figure medium scale is 20mm).

[Fig. 4 A] Fig. 4 A shows the wavelength (nm) of the ultraviolet-visible absorption spectroscopy in blue gold nano grain of the present invention synthetic and the relation between the absorbance.

[Fig. 4 B] Fig. 4 B show each reaction temperature in blue gold nano grain of the present invention in Fig. 4 A synthetic (℃) and maximum absorption wavelength between relation.

[Fig. 5] Fig. 5 shows the relation between the concentration of the maximum absorption wavelength (nm) of the ultraviolet-visible absorption spectroscopy in blue gold nano grain of the present invention synthetic and ascorbic acid.

[Fig. 6] Fig. 6 shows the comparison of the detection sensitivity when blue gold nano grain of the present invention is used as immunochromatography reagent.

The specific embodiment

As blue gold nano grain of the present invention, although it is desirable in a step, produce the big blue gold nano grain of those average grain diameters, yet, at first form the particle of pre-sizing, carry out growth step then and be only reasonably with the particle that obtains greater particle size.Blue gold nano grain of the present invention is that the gold nano grain of 20nm to 200nm constitutes by average grain diameter.The average grain diameter of the colloid gold particle by gold nano grain of the present invention being scattered in the colloidal solution that obtains in the medium is 20nm to 200nm, is preferably 40nm to 180nm, most preferably is 50nm to 120nm usually, and optimum is 60nm to 100nm.Various angles the practical applications such as remarkable identity during from inspection consider that described gold nano grain preferably has the shape of the flat sugared shape of gold of sharp-pointed particle diameter distribution and homogeneous.Average grain diameter can be determined (colloidal solid that makes the colloidal sol shape is handled with 14000 to 5530000 * g speed rotation and to it, determines average grain diameter by the settling rate of this colloidal solid) usually by the gravity light scattering method in ultracentrifuge.In the present invention, to from by transmission electron microscope (TEM, " JEM-2010 ", JEOL, Ltd. product) area diameter projected of 100 particles selecting at random in the projection print of Pai Sheing is measured, and determines the average grain diameter (average particulate diameter) based on mean value then.

When (for example setting X-axis; the size of gold nano grain) and Y-axis (for example; the number ratio) distributes with the particle diameter of between them, drawing gold nano grain and during the distribution curve of average grain; the summit of the distribution curve of gold nano grain of the present invention is located substantially on the particle size range of 40nm to 120nm usually; be preferably 50nm to 110nm, be more preferably 60nm to 100nm.This has disclosed this distribution curve relative narrower, and this represents that a lot of nano particles have close particle diameter, thereby has homogeneous particle diameter.Can expect that this nano particle shows stable and behavior highly reliably, and suppress the generation of the error span that causes owing to the foreign matter of sneaking into wherein.

Quantitatively, the gross weight that belongs to the gold nano grain in 20nm to the 200nm scope is generally more than 40%, is preferably more than 60%, is more preferably more than the 80 weight %.Particle, spherical particle and unreacted residue that remaining part left behind by there not being growth constitute.

Gold nano grain of the present invention is the flat sugared shape nano particle of so-called gold with nuclear and three-dimensional protrusion.Those gold nano grains that have any average grain diameter in the scope of 20nm to 200nm can obtain by the operation that changes manufacture method.As marking particle the time, aspect the accuracy that improves the visual judgement of carrying out based on the particular color of marking particle, the gold nano grain that those average grain diameters fall into (preferably in the scope of 55nm to 100nm) in the scope of 50nm to 120nm is excellent.

The flat sugared shape particle of these gold preferably has a plurality of three-dimensional protrusion.The term average grain diameter of Shi Yonging represents to comprise described nuclear projection and definite value herein.Each nuclear of gold nano grain of the present invention has about 1 to 20 projection, and preferably each nuclear has about 4 to 10 projections.The number of these projections or length are difficult to pre-determine because depending on the growth of nuclear very much.

The gold nano grain and the collaurum nano particle that have three-dimensional protrusion on the nuclear are referred to as the flat sugared shape gold nano grain of grafting shape, multiway shape or gold and collaurum nano particle respectively.As so-called gold nano grain and collaurum nano particle; they can be for having the various structures of three-dimensional protrusion; and call by known titles such as nanocube, nanometer rods, nanometer pin (nanopods), star gold nano grain or grafting shape gold nano grains; wherein said grafting shape gold nano grain has the nuclear shown in Figure 1A and 1B, grows baculum with three dimensional constitution on this nuclear.Colour developing has shape or the structure that is similar to for the tetrapod of breakwater for the collaurum nano particle of distinct blueness.Therefore, adopted this term, the collaurum nano particle that will have a branch of the grafting of being grown to is called " single pin ", and the collaurum nano particle can have different shape along with the increase of number of branches, as " both feet ", " tripod ", " four pin " and " five pin ".In the present invention, the protrusions number of each nuclear is preferably relatively large, thereby this shape is referred to as " multiway ".

Be rendered as red spherical shape colloid gold particle and compare with traditional, the color that presents according to multiway shape colloid gold particle of the present invention or the flat sugared shape colloid gold particle of gold depends on their expansion mode.This makes the collaurum nanoparticles solution present to comprise blue multiple color.

Particularly, as the blue gold nano grain of the present invention of the grafting shape that typically has three-dimensional protrusion, multiway shape or the flat sugared shape of gold, show have shape shown in Figure 1A or Figure 1B gold nano grain as an example.These gold nano grains heart therein partly have so-called nuclear, and grow projection or branch as grafting at nuclear.Because the growth starting point of grafting closely contacts with described nuclear, so they look like the flat sugared shape gold nano grain of projection and the integrated multiway shape gold nano grain of nuclear or gold.

The example of the gold nano grain among Figure 1A and Figure 1B has specifically illustrated the example of the blue gold nano grain that is of a size of about 50nm.More specifically, the gold nano grain shown in Figure 1A and Figure 1B has the average grain diameter (DLS) of 66.5nm and the maximum absorption wavelength of about 610nm.In addition, according to the measurement by tem observation, the mean outside diameter of gold nano grain is that 62.2nm, average core diameter are that 35.7nm, average projection length are that 13.2nm and projection angle are about 50 degree.The AR(aspect ratio of gold nano grain) be more than 1.Self-evident is to change mean outside diameter, average core diameter, average projection length and the projection angle of gold nano grain of the present invention by the product of considering different colours arbitrarily.

In the present invention who comprises embodiment, wavelength is measured by following mode.Use ultraviolet-visible absorption spectroscopy instrument (spectrometer name: " UV-2550 ", the product of Shimadzu Corporation) to measure wavelength.Measure and carry out under the following conditions: quartz cell: 10mm; Wavelength: 800-200nm; Bandwidth: 0.5nm.

Blue gold nano grain and blue collaurum nano particle are effective to the exploitation of multinomial diagnosticum.When existing many to judge lines, described blue gold nano grain and blue collaurum nano particle can be eliminated the possibility of visual mistaken diagnosis when judging.The gold nano grain that is used as the immunologic assay mark substance in this multinomial diagnosticum is the immunologic assay mark substance with following feature: it is made of at least two kinds of variform gold nano grains.More specifically, suited by at least two kinds of immunologic assay mark substances that constitute in the flat sugared shape blueness gold nano grain of spherical red gold nano grain and the grafting shape with three-dimensional protrusion, multiway shape or gold.

The gold nano grain of the present invention that is used as the immunologic assay mark substance in the multinomial diagnosticum comprises the mixture of (for example) two or three variform gold nano grain (hereinafter being referred to as " mixture type gold nano grain mark substance "), for example spherical gold nano grain and the mixture with gold nano grain of three-dimensional protrusion.In this case, when analyzing the particle diameter distribution according to shape, described mixture type can form the distribution curve with two summits, i.e. the grading curve that is formed by spherical gold nano grain and distributed by the particle diameter that the gold nano grain with three-dimensional protrusion forms.Self-evidently be for by having the mixture that gold nano grain that three kinds of shapes differ from one another forms, can draw out the grading curve with three summits.In the present invention, if do not consider shape difference the particle diameter of at least two kinds of metal nanoparticles is not analyzed, then because the relatively large gold nano grain of the less relatively gold nano grain of average grain diameter and average grain diameter exists with the form of mixture, therefore average grain diameter is fallen in the wide relatively scope of 20nm to 100nm.In a word, when each grading curve formed sharp-pointed peak, the amount of the gold nano grain that expression is predetermined was bigger, therefore can improve measurement accuracy.To the object lesson of this mixture type gold nano grain mark substance be described below.

Specifically describe the state of " mixture type gold nano grain mark substance " of the present invention.When the mixture type gold nano grain mark substance of invention is identified as two kinds, for example, a kind of is spherical gold nano grain, and another kind is the gold nano grain with three-dimensional protrusion, consider the detection sensitivity of mark, assert that mixture contains this two kinds of particles with the quality % of 10:90-90:10.This expression, when the amount of spherical gold nano grain was 40 quality %, the amount with gold nano grain of three-dimensional protrusion was 60 quality %.Self-evidently be that calculating is by removing unreacted matters, not having other materials outside the expectation materials such as nano particle that growth left behind and impurity to carry out.

For example, the spherical gold nano grain that constitutes this mixture type gold nano grain mark substance is relatively large particle, and its average grain diameter is 20nm to 220nm, is preferably 30nm to 200nm, is more preferably about 40nm to 150nm.As for the gold nano grain with three-dimensional protrusion, those average grain diameters are that the gold nano grain of about 20nm to 200nm may reside in the mixture.Stability, mark accuracy and reliability for the distinctiveness that improves color, long color stability, colloid, described average grain diameter is preferably 40nm to 180nm, usually most preferably be 50nm to 120nm, and optimum scope is 60nm to 100nm.

Mixture type gold nano grain mark substance can obtain spherical gold nano grain mark substance and the gold nano grain with three-dimensional protrusion by (for example) with the straightforward procedure that predetermined ratio mixes, wherein said spherical gold nano grain mark substance is previously prepared, and has predetermined average grain diameter.

Contain at least two kinds of gold nano grains as mixture type gold nano grain mark substance of the present invention and by the immunization certification mark material that at least two kinds of variform gold nano grains constitute, these two kinds of gold nano grains are used as the mark substance that constitutes labelled reagent, described labelled reagent is modified the mensuration material of can binding immunoassay learning the target substance in the mensuration system, and carry out mark by being combined with this target substance, wherein

1) average grain diameter of described two kinds of gold nano grains is 20nm to 220nm, and

2) a kind of in described two kinds of gold nano grains is spherical, and another kind has at least four three-dimensional protrusion.

When using this mixture type gold nano grain mark substance, can clearly distinguish various antigens by the difference of colors such as red and blueness.Therefore, can alleviate the on-the-spot inspection burden of medical treatment and simplification and check operation.Therefore can obviously improve its practicality.

Colloid gold particle of the present invention is rendered as blueness when visual observation.This expression is by colloid gold particle being scattered in the color that the colloidal gold solution that obtains in the water equal solvent is rendered as blueness when the visual observation or is similar to blueness, as blue-green or bluish violet.More specifically, this expression, the form and aspect of described solution are defined as 3P to 1P, 10PB to 1PB, 10B to 1B, 10BG to 1BG or 10G to 8G by Munsell colour system.Wherein, consider the differentiation with redness, the form and aspect of preferred 10PB to 1PB, 10B to 1B or 10BG to 1BG.About colorimetric method, fill the quartz cell (optical path length: about 10mm) that is used for spectrophotometry with colloidal solution, go up visual its tone of determining in white background (white print drawing paper), based on commercially available munsell color atlas described form and aspect are estimated then.

Manufacturing comprises according to the method for gold nano grain of the present invention: in the nuclear shape stage, wherein, with first reducing agent first gold medal salt in the aqueous solution is reduced to the flat sugared shape nuclear gold nano grain of gold; And growth phase, wherein, drip the second gold medal salt and second reducing agent simultaneously, so that being grown to, described nuclear gold nano grain has the flat sugared shape gold nano grain of larger sized gold.Described growth phase can carry out at least one times.

In order to form the flat sugared shape gold nano grain of the gold with longer projection at growth phase, used the mixture of second reducing agent and first reducing agent, that is, used the organic acid that contains piperazine ring as Good ' s buffer solution component.

According to the concentration of second reducing agent that uses in the growth phase, the amount of first reducing agent that is used in combination with second reducing agent is no better than the amount of second reducing agent.Namely making in the aqueous solution of nuclear gold nano grain growth at growth phase, is 0.01mM-100mM with the concentration adjustment of employed first reducing agent.

For the behavior of the chemical species of analyzing blue gold nano grain, as an embodiment, the particle that will be equivalent to the nuclear particle before the growth response is called " particle 1 ", and by mixing the AuCl of 0.43mM ₄The solution for preparing " particle 1 " with the HEPES of 39.0mM.The particle that will be equivalent to the particle of growing after the growth response is called " particle 2 ", and by mixing the AuCl of 0.05mM ₄, the HEPES of 0.82mM and 0.10mM ascorbic acid prepare the solution of " particle 2 ".Behavior to gained solution is analyzed.

The example that increases particle diameter of the present invention based on Fig. 2 A and 2B to not changing peak wavelength describes.In the absorption spectrum of Fig. 2 A " particle 1 ", the present inventor has realized making under the situation of the peak wavelength that does not change ultraviolet-visible absorption spectroscopy particle diameter to be grown to " particle 2 " among Fig. 2 B in the present invention.In Fig. 2 A and 2B, peak wavelength is represented the scope of about 570nm to 630nm.

Being used for karyomorphism of the present invention becomes the example of the first gold medal salt in stage to comprise: gold chloride, gold tribromide, three are fluoridized gold, gold triiodide, gold tricyanide, aurous chloride, aurous iodide, are fluoridized inferior gold, aurous cyanide, hydroxyl oxidize gold (hydroxy gold oxide), three nitric acid gold (gold trisnitrate) and nitric acid gold salt and hydrates thereof such as (gold nitrate); And the wang aqueous solution of gold.Gold salt is not limited to above-mentioned substance, but can use any material that can form the first gold medal salt in the aqueous solution.

Become first reducing agent in stage as being used to karyomorphism of the present invention, can use the organic acid that contains piperazine ring as Good ' s buffer solution component.Its example includes but is not limited to 2-[4-(2-ethoxy)-1-piperazinyl] ethyl sulfonic acid (below will abbreviate " HEPES " as), 4-(2-ethoxy)-1-piperazine propane sulfonic acid (below will abbreviate " HEPPS " as), 4-(2-ethoxy) piperazine-1-(2-hydroxy propane-3-sulfonic acid) (below will abbreviate " HEPPSO " as), piperazine-1, two (2-ethanesulfonic acid) (below will abbreviate " PIPES " as) of 4-, 3-[4-(2-ethoxy)-1-piperazinyl] propane sulfonic acid (below will abbreviate " EPPS " as) and piperazine-1, two (2-hydroxyl-3-N-morpholinopropanesulfonic acid) (below will abbreviate " POPSO " as) of 4-.As reducing agent, more preferably HEPES, HEPPSO and PIPES.More preferably HEPES is as reducing agent.Can optionally use the combination of these reducing agents.

As the second gold medal salt that in growth phase of the present invention, uses, can use to be described as the example that karyomorphism becomes the employed first gold medal salt in the stage.The second gold medal salt and the first gold medal salt can be identical or different.Can preferably gold chloride be used as the first gold medal salt and the second gold medal salt.

As second reducing agent that in growth phase of the present invention, uses, can use the organic acid with reproducibility, as ascorbic acid and derivative, citric acid and derivative thereof, alpha-hydroxy carboxylic acid compounds (as D (L)-malic acid, D (L)-tartaric acid, hydroxymalonic acid and glutinous acid), lactic acid, tannic acid and reduced sugar.Wherein, preferred ascorbic acid and derivative and citric acid and derivative thereof, wherein most preferably ascorbic acid and derivative thereof.Also can use their mixture.

As ascorbic acid and derivative thereof, can use those ascorbic acid with reproducibility and derivatives thereof, as ascorbic acid (its salt), its isomers or analog and derivative thereof.Example comprises L(or D)-alkali salt of ascorbic acid such as the alkali metal of ascorbic acid such as ascorbic acid Arrcostab, sodium ascorbate such as halo deoxidation ascorbic acid, ascorbic acid ethyl ester such as ascorbic acid, arabo-ascorbic acid, erythritic acid (erythorbic acid), scorbamic acid (scorbamic acid), dehydrogenation arabo-ascorbic acid, deoxidation ascorbic acid, chloro deoxidation ascorbic acid and Calcium Ascorbate.Wherein, preferred especially L(or D)-ascorbic acid (its salt) and arabo-ascorbic acid.Also can optionally use their mixture.

As citric acid and derivative thereof, can use citric acid and derivative thereof with reproducibility, as citric acid (its salt), its isomers or analog and derivative thereof.Its example comprises alkali salt and citric acid Arrcostabs such as methylcitrate and ETHYL CTTRATE such as ammonium salts such as alkali metal salt, ammonium citrate, calcium citrate such as citric acid, isocitric acid, citric anhydride, isocitric acid acid anhydride, natrium citricum and potassium citrate.Wherein, special optimization citric acid and natrium citricum.Also can optionally use their mixture.

It is 0-40 ℃ that karyomorphism of the present invention becomes the reaction temperature in the stage, is preferably 10 ℃-30 ℃ (room temperatures), is more preferably 15 ℃-25 ℃.Reaction was carried out 30 minutes to 5 hours.Reaction temperature surpasses 40 ℃ of numbers that can increase spherical particle, and reduces productive rate.Even reaction temperature is reduced to less than 0 ℃ productive rate is increased, thereby be nonsensical technically, and uneconomical and waste.

Become the stage to be formed with in the aqueous solution of nuclear gold nano grain at karyomorphism, it is 1mM to 150mM that karyomorphism of the present invention becomes the concentration of employed first reducing agent in the stage, is preferably 30mM to 100mM.When this concentration during greater than 150mM, this concentration has surpassed necessary concentration and has become meaningless technically, and uneconomical and waste.When this concentration during less than 1mM, the function of reducing agent too a little less than, make that being not enough to carry out karyomorphism becomes reaction.

Become the stage to be formed with in the aqueous solution of nuclear gold nano grain at karyomorphism, it is 0.1mM to 100mM that karyomorphism of the present invention becomes the concentration of the employed first gold medal salt in the stage, is preferably 1mM to 50mM, is more preferably 5mM to 25mM.

The mmol/L of the term of Shi Yonging " mM " expression herein.

React by this way, make to become in the stage at karyomorphism, by making first reducing agent with above-mentioned concentration range and having in the colloidal gold solution that the first gold medal reactant salt of above-mentioned concentration range obtains, the concentration of gold falls in the scope of 0.1mM to 100mM.

Reaction temperature in the growth phase of the present invention is 0 to 40 ℃, is preferably 10 ℃ to 30 ℃ (room temperature), is more preferably 15 ℃ to 25 ℃.Reaction was carried out 1 hour to 10 hours.When reaction temperature surpassed 40 ℃, particle tended to the englobement particle, thereby caused productive rate to reduce.Simultaneously, therefore the maximum absorption wavelength of ultraviolet-visible absorption spectroscopy migrates to the shorter wavelength side less than 570nm.Reaction temperature is reduced to less than 0 ℃ does not have effect, is skimble-skamble therefore.

When from the angle of the amount that reduces unreacted gold chloride the reasonable synthetic method of gold nano grain of the present invention being furtherd investigate, will describe based on Fig. 4 A and the result of 4B.By the relation between unreacted gold chloride and reaction temperature or the reactivity is studied, found that, be set to low temperature by reaction temperature, make nano particle and show the behavior that becomes more blue.Fig. 4 A and 4B have disclosed reaction temperature, and to be set to 10 ℃ to 35 ℃ be only.

Same, when describing based on Fig. 4 A and 4B, Fig. 4 A shows under the differential responses temperature of 10 ℃, 20 ℃, 30 ℃ and 40 ℃ the result of study for wavelength (nm).When reaction temperature is set to more than 40 ℃, can observes from blueness and migrate to red trend.When so-called reaction temperature increased, it is redder that the collaurum nano particle trends towards becoming.On the other hand, when described reaction temperature reduced, it is more blue that the collaurum nano particle trends towards becoming.More specifically, can find from Fig. 4 B, be set to 10 ℃ to 30 ℃ by reaction temperature, optimal is to be set to 15 ℃ to 25 ℃, can easily obtain the colloid gold particle that maximum absorption wavelength is about 600nm.

Growth has in the aqueous solution of nuclear gold nano grain in growth phase, and the concentration of second reducing agent such as employed ascorbic acid or derivatives thereof is 0.01mM to 100mM in the growth phase of the present invention, is preferably 1mM to 50mM, is more preferably 5mM to 25mM.

Fig. 5 shows the measurement result of variation of maximum absorption wavelength of the ultraviolet-visible absorption spectroscopy of colloid gold particle suspension, and wherein this colloid gold particle suspension is to obtain by the use amount that changes ascorbic acid in the growth phase described in the embodiment 4.Draw the weight concentration of the aqueous ascorbic acid that adds in the growth phase along the abscissa of Fig. 5.To consider the optimum amount of ascorbic acid or derivatives thereof in the growth phase, disclose as Fig. 5, to be the blueness of collaurum in order developing the color, the aqueous ascorbic acid of interpolation can be with 0.02 to 0.07(quality %) wideer relatively concentration range use.Yet from considering that with the angle of the relation of blue wavelength growth has in the whole aqueous solution of nuclear gold nano grain in growth phase, the optimum condition of the concentration of ascorbic acid is 0.075mM to 0.15mM.According to the present inventor's discovery, this scope is criticality technically.

Growth has in the aqueous solution of nuclear gold nano grain in growth phase, and the concentration of the second gold medal salt that uses in the growth phase of the present invention is 0.1mM to 100mM, is preferably 0.2mM to 20mM.

The addition of second reducing agent that uses in growth phase of the present invention can be 5 times to 500 times of molar concentration of the nuclear gold nano grain that adds, and is more preferably 25 times to 250 times.The addition of the second gold medal salt that uses in growth phase of the present invention can be 0.1 times to 10 times of molar concentration of the nuclear gold nano grain that adds, and is more preferably 0.5 times to 5 times.

The second gold medal salt and second reducing agent are added drop-wise in the colloidal gold solution synthetic in the nucleus growth stage simultaneously, its drop rate is 0.1ml/ minute to 3.0ml/ minute, be preferably 0.3ml/ minute to 1.5ml/ minute, be preferably 0.5ml/ minute to 1.0ml/ minute especially.

Immunologic assay method according to the present invention is based on the determination method of immunology specific binding reaction, and wherein said immunology specific binding reaction comes from the compatibility that biomolecule has.For example, known have immunostaining, agglutinating reaction, ELISA and an immunochromatographic method.As this combination that comes from compatibility, Ag-Ab is in conjunction with representative and be widely used in the immunologic assay method.Be not only this combination, also can use sugar-agglutinin combination, hormone-receptors bind, enzyme-inhibitor in conjunction with, nucleic acid-complementary nucleic acid in conjunction with or nucleic acid and have can with the combination of proteins of its combination.As immune response or immunological response, available have (for example) sandwich method or a competition law, in sandwich method, formed the sandwich compound of for example " solid matrix antibody-antigen-labelled antibody (labelled antigen) " to catch and to detect antigen, the competition ratio juris is the competitive reaction that utilizes insolubilized antibody and the free antigen in the corpse or other object for laboratory examination and chemical testing and add the labelled antibody (labelled antigen) of the scheduled volume in the reaction system to.Wherein, utilize the method the most easily of the sandwich reaction between antigen and the antibody to be to use the immunochromatographic method of chromatography.Because immunochromatographic method is simple to operate, only need the minute of lacking very much and help visual judgement, thereby it is the people institute method of employing usually.

Based on Fig. 6, the superiority of the detection sensitivity of blue gold nano grain of the present invention when being used for various immunochromatography reagent is illustrated.Fig. 6 show with check like the immunochromatography detection type of embodiment 8 described Type B influenza viruses, by using the measurement result of the color intensity that immunochromatographiassays assays instrument (immunochromatographic reader) obtains." particle 1 " is to become blue colloid gold particle suspension that the stage forms as the system of mark substance the karyomorphism by embodiment 1 only, and " particle 2 " is to become blue colloid gold particle suspension that stage and growth phase form as the system of mark substance the karyomorphism by embodiment 1.As antigen, in the situation of " particle 1 ", after being diluted to 1400 times, uses the aqueous solution that will contain 60 μ g/ml antigens, in the situation of " particle 2 ", after being diluted to 2400 times, uses the aqueous solution that will contain 60 μ g/ml antigens.With " particle 1 " (antigen diluent multiplying power: 1400 times) and " particle 2 " (antigen diluent multiplying power: 2400 times) when comparing, the result shows in bright gay color in " particle 2 ", infers that this is because the surface area of " particle 2 " is bigger.Owing to there being numerous reasons, therefore can not judge its accurate reason.Yet, the detection sensitivity excellence of blue gold nano grain of the present invention, and by using immunochromatography reagent to have the effect of the accuracy of the visual judgement of obvious raising.

In immunologic assay method of the present invention, the sample (corpse or other object for laboratory examination and chemical testing) (for example) that contains detected object is mainly the organism sample, as the extract of blood, serum, blood plasma, urine, saliva, marrow liquid, sweat, tears, amniotic fluid, nipple juice, nasal mucus, phlegm, nasal cavity or throat swab liquid, skin secretion and tissue, cell or ight soil.

Detected object of the present invention has no particular limits, maybe can prepare such material with material that its specificity is combined (for example, as the reaction between antigen and the antibody or nucleic acid and the material of being combined with the specificity in the reaction between its complementary nucleic acid) and get final product as long as exist.Described detected object can be itself to have antigenic comlete antigen, or itself does not have antigenicity but can obtain antigenic haptens (incomplete antigen) by chemical modification.Maybe can prepare the material of being combined with these detected object specificitys as long as exist.This material can be monoclonal antibody or polyclonal antibody.

The example of the detected object among the present invention comprises: peptide hormone (growth hormone (GH), corticotropin (ACTH), melanocyte stimulating hormone (MSH), lactogen, thyrotropic hormone (TSH), luteinising hormone (LH), follicle-stimulating hormone (FSH) (FSH), pituitrin, the Calcium Metabolism Regulation hormone, the kidney hormone, gastrointestinal hormone, vasoactive hormone and human chorionic gonadotrophin placental hormones such as (hCG)), PAP (PAP), prostate specific antigen (PSA), alkaline phosphatase, transaminase, trypsase, pepsin, alpha-fetoprotein (AFP), carcinomebryonic antigen tomour specific materials such as (CEA), immunoglobulin G serum protein components such as (IgG), rheumatoid factor, thrombocytin, urokinase, ferritin, the P material, estrogen (as estrone), fecal occult blood, syphilis antibody, influenza virus, adenovirus, RS virus, rotavirus, HBs antigen, HBs antibody, CHLA Casset, bacterial antigens such as micrococcus scarlatinae (Streptococcus pyogens) antigen, natural or synthetic progestational hormone, male sex hormones such as testosterone, cortex hormone of aadrenaline such as cortisol, other steroids is (as cholesterol, bile acid, cardiac stimulant sex steroid and sapogenin etc.), adrenaline, dopamine, the physiologically active alkaloid, contain amino psychotropic agent, small-molecular peptides such as TRH, thyroid hormones such as diiodothyronine, prostaglandin, vitamins, antibiotic such as penicillin, DNA, RNA, oligonucleotides, polynucleotides, their amplified production, component in other body, and offer medicine and metabolite in the body, food is (as pork, beef, chicken and egg etc.) and food (comprise pork, beef, chicken and egg etc.) extract.In these detected objects, preferred virus is more preferably influenza virus, adenovirus and RS virus.

In the present invention, an only corpse or other object for laboratory examination and chemical testing is nasal mucus, nasal cavity or throat swab liquid or phlegm.By using developping solution to dilute a this corpse or other object for laboratory examination and chemical testing in advance, can detect definitely as the collection of the detected object antigen (virus: mainly be influenza virus, adenovirus, RS virus) from the respiratory disorder patient.

In the present invention the immunochromatography of Shi Yonging with developping solution normally by buffer, salt, blocking agent and nonionic surface active agent prepare as solvent and to wherein adding with water.Interpolation has no particular limits in proper order, and can add simultaneously.When using developping solution, the mixture of detected sample (target sample) and developping solution can be supplied with/is added drop-wise on the sample pad (sample interpolation part) and launch.Difference per sample also can be supplied with detected sample earlier/be added drop-wise on the sample pad (sample interpolation part), then developping solution is supplied with/is dripped in sample pad (sample interpolation part) and go up to launch sample.

The buffer that is used for immunochromatography developping solution of the present invention has no particular limits, as long as its effect (cushioning effect) is not because the evaporation of the change in concentration of sample due to adding, sample or dilution or some come from the sneaking into of foreign matter of outside is subjected to having a strong impact on.

The example of the buffer among the present invention comprises the Good buffer solution, as acetate buffer (acetic acid+sodium acetate), phosphate buffer (phosphoric acid+sodium phosphate), citrate buffer solution (citric acid+natrium citricum), borate buffer, tris-HCl buffer solution (trishydroxymethylaminomethane+hydrochloric acid), TE buffer solution (tris+ ethylenediamine tetra-acetic acid), TAE buffer solution (tris+ acetic acid+ethylenediamine tetra-acetic acid), tbe buffer liquid (tris+ boric acid+ethylenediamine tetra-acetic acid) and HEPES buffer solution (2-[4-(2-ethoxy)-1-piperazinyl] ethyl sulfonic acid).Wherein, preferred acetate buffer, phosphate buffer and tris-HCl buffer solution are more preferably the tris-HCl buffer solution.

Be used for immunochromatography of the present invention and have no particular limits with the salt of developping solution, so long as the salt that the reaction by bronsted lowry acids and bases bronsted lowry obtains gets final product.Its example comprises sodium chloride and potassium chloride.Preferred sodium chloride wherein.

Comprise for the examples of nonionic surfactants of immunochromatography of the present invention with developping solution: polyoxyethylene alkyl ether, the polyoxyethylene/polyoxypropylene alkyl ether, polyoxyethylene sorbitan fatty acid ester (" Tween " series, trade name, the product of Sigma Aldrich), polyoxyethylene is to uncle's octyl phenyl ether (" Triton " series, trade name, the product of Sigma Aldrich), polyoxyethylene is to uncle's nonylplenyl ether (" Triton N " series, trade name, the product of Sigma Aldrich), alkyl polyglucoside, fatty diglycollic amide and alkyl list glyceryl ether etc.These nonionic surface active agent can use separately, perhaps use with their form of two or more mixtures.

Use in the developping solution at immunochromatography of the present invention, add more than one suppress the side reaction that is caused by bioaffinity or the known additive that suppresses nonspecific reaction be possible and effectively, described known additive be (for example) as the protein of the blocking agent of the promoter of antigen-antibody reaction or inhibition nonspecific reaction (as bovine serum albumin, gelatin and casein etc.), macromolecular compound is (as polyethylene glycol, methylcellulose, PVP, polyvinyl alcohol, glucan etc.), and ionic surfactant or polyanion are (as dextran sulfate, heparin, polystyrolsulfon acid, chondroitin sulfate etc.), perhaps antibiotic.Their adding can not influence effect of the present invention.Will for promote antigen-antibody reaction or suppress protein, macromolecular compound, ionic surfactant or the polyanion of nonspecific reaction or more than one of antibiotic remain on the migration path of the mobile phase on the chromatography media that constitutes fixing phase also be possible and effective.Their maintenance does not influence effect of the present invention.

In the device for immunochromatography for detection of the detection target in the corpse or other object for laboratory examination and chemical testing, its structure and operation/detection method are known.Device for immunochromatography has (1) sample addition portion, (2) mark substance maintaining part, (3) chromatography media, (4) test section (being also referred to as " detection unit "), (5) absorbent portion and (6) base plate usually.

Use developping solution, the corpse or other object for laboratory examination and chemical testing sample drop that dilutes a corpse or other object for laboratory examination and chemical testing in advance and obtain is added on the sample pad of conventional device for immunochromatography, its direction along absorbent portion in the immunochromatography medium is launched, thereby antigen-antibody reaction is taken place.Based on this reaction, can identify or inspection such as judgement the detection target in the corpse or other object for laboratory examination and chemical testing.

To describe device for immunochromatography below.

Sample addition portion (1) is made by porous chipses such as glass filter paper, and porous chipses such as described glass filter paper allow the rapid absorption of sample, but a little less than the confining force, make sample move to reactive site rapidly.

Mark substance maintaining part (2) maintains utilizes marker components reagent component to be carried out mark and the labelled reagent that obtains.The example of marker components comprises: colloidal metal particle, latex particle, enzyme and fluorescent chemicals.Wherein, the colloidal metal particle is best suited for.The colloidal solid cup of blue gold nano grain of the present invention is as marker components.Reagent component is particle or the molecule with ability of discriminance analysis thing, preferred monoclonal antibody or polyclonal antibody, perhaps their fragment (second reagent).

Chromatography media (3) has test section (4) at membrane carrier.Membrane carrier has no particular limits, and gets final product so long as can absorb a sample corpse or other object for laboratory examination and chemical testing and move it by capillarity.For example, the group that constitutes of the optional free nitrocellulose of membrane carrier, cellulose acetate, nylon, polyether sulfone, polyvinyl alcohol, polyester, glass fibre, polyolefin, cellulose and the man-made polymer that made by their composite fibre.

In test section (4), monoclonal antibody or polyclonal antibody or its fragment (first reagent) support and are fixed on the nitrocellulose chip.

Absorbent portion (5) is made by the material (for example glass filter paper etc.) of the ability with the superfluous sample of rapid absorption.

Base plate (6) is base material.Distinguish coating adhesive or Continuous pressing device for stereo-pattern by a face at base plate, make base plate have cohesive at this face, part or all of sample addition portion (1), mark substance maintaining part (2), chromatography media (3), test section (4) and absorbent portion (5) is combined on the adhesive surface of base plate securely.Base plate (6) has no particular limits as base material, so long as make it have impermeability to sample liquid or non-poisture-penetrability gets final product by adhesive.

Can be monoclonal antibody or polyclonal antibody for detection of the reagent component (first reagent) of portion (4) and the one or both that is used for the reagent component (second reagent) of labelled reagent.Consider that from the angle of measuring sensitivity etc. the reagent component (second reagent) that is used for labelled reagent is preferably the high monoclonal antibody of specificity.Reagent component (first reagent) for detection of portion (4) namely can be that monoclonal antibody also can be polyclonal antibody.

Monoclonal antibody and polyclonal antibody and their fragment are known and available.And can be prepared by known method.The examples of animals that produces antibody comprises people, mouse, rat, rabbit and sheep etc.As immunoglobulin (Ig), can use any one among IgG, IgM, IgA, IgE and the IgD.

Monoclonal antibody can obtain by conventional method.Will be through spleen cell and myeloma cell's hybridization of antigen (for example A type influenza virus) mice immunized.Select to produce the hybridoma of target antibody, obtain the monoclonal antibody by its generation.For example can reference

The method of delivering with Milstein (Nature, 256 (1975), 495-497).

Polyclonal antibody can pass through conventional method, utilize antigen (for example A type influenza virus) that the animal (for example people, mouse, rat, rabbit, sheep or horse etc.) that can produce antibody is carried out immunity and obtain antiserum, separation obtains target antibody and obtains in the antiserum of gained.

Although as described in embodiments of the invention: as the reagent component (second reagent) that is used for labelled reagent, used the anti-A type influenza monoclonal antibody that comes from mouse, and as the reagent component (first reagent) for detection of portion (4), used the anti-A type influenza monoclonal antibody that comes from mouse, but reagent component is not limited to this.Also can use the anti-A type influenza polyclonal antibody that comes from mouse.

Be the summary of resolution principle below.

1. the corpse or other object for laboratory examination and chemical testing sample (with the corpse or other object for laboratory examination and chemical testing of developping solution dilution) with ormal weight (common 0.1 to 2ml) drips on sample pad (1).When dripping corpse or other object for laboratory examination and chemical testing sample, though corpse or other object for laboratory examination and chemical testing sample is absorbed rapidly in sample pad (1), the gained sample pad together begins mobile with sample immediately.When utilizing immunochromatography with reagent composition impregnation sample pad (1), immunochromatography is dissolved in the moisture of corpse or other object for laboratory examination and chemical testing sample with reagent composition, and together begins mobile with corpse or other object for laboratory examination and chemical testing sample.

2. corpse or other object for laboratory examination and chemical testing sample is at first mobile to mark substance maintaining part (2).When corpse or other object for laboratory examination and chemical testing sample passed through this position, the labelled reagent that keeps on the mark substance maintaining part (2) (second reagent) was dissolved in the moisture of sample, and together mobile with sample.

3. then, be dissolved in labelled reagent in the corpse or other object for laboratory examination and chemical testing sample moisture by the test section (4) on the chromatography media (3).Herein, the immunochromatography reagent composition that is dissolved in the corpse or other object for laboratory examination and chemical testing sample has suppressed the non-specific binding reaction.When corpse or other object for laboratory examination and chemical testing sample contains detected object (for example antigen), because antigen and antibody specific association reaction, it is to be clipped in mode and this antibody generation specific reaction and the combination between antibody and the labelled reagent, make test section (4) painted thus, described antibody supports and is fixed on the test section (4).When corpse or other object for laboratory examination and chemical testing sample did not contain detected object (for example antigen), labelled reagent was dissolved in the moisture of sample, even sample the specificity association reaction can not take place by the test section (4) on the chromatography media (3) yet.Therefore, test section (4) is not colored.

4. last, the moisture of sample moves to absorbent portion (5).

Can whether make correct judgement to the existence of the detected object in the corpse or other object for laboratory examination and chemical testing sample (for example antigen) thus.

Below will the present invention will be described in detail by embodiment and Comparative Examples.Yet the present invention is not limited to or is subject to these embodiment.

(i) measurement of average grain diameter

Although can determine that average grain diameter (handle with 14000 to 5530000 * g speed rotation and to it by the colloidal solid that makes the colloidal sol shape in ultracentrifuge by the gravity light scattering method usually, settling rate by this colloidal solid is determined average grain diameter), however use dynamic light scattering (DLS) analyzer to calculate average grain diameter in the present invention.Also can be to from by transmission electron microscope (TEM, " JEM-2010 ", JEOL, the product of Ltd.) area diameter projected of 100 particles selecting at random in the projection print of taking is measured, and calculates the average grain diameter (average particulate diameter) based on mean value.Same, by the mean value calculation average core size of the area diameter projected of 100 particles that are selected from the TEM projection print at random, calculate average projection length (average length of grafting) with the difference of average grain diameter and average core size divided by 2.

[embodiment 1]

In this embodiment, become in the stage at karyomorphism, the HEPES reduction by being used as first reducing agent is as the gold chloride of the first gold medal salt, thereby forms the flat sugared shape colloid nuclear of gold.Then, in the stage of growth, drip simultaneously as the gold chloride of the second gold medal salt with as the L-ascorbic acid of second reducing agent, have the flat sugared shape collaurum of gold of bigger particle diameter with formation.

[karyomorphism becomes the stage]

With 10ml HEPES(4 * 10 ^-2Mol/L pH7.8) injects the glass container with cover of 10ml, holds it in temperature control and becomes 25 ℃ until fluid temperature in bathing.In addition, with 0.7g(1.6 * 10 ^-2Mol) four hydration gold chlorides are dissolved in the ultra-pure water of 100ml.Gained solution remained on ice become 4 ℃ until fluid temperature.When the fluid temperature of the aqueous solution of the aqueous solution of HEPES and gold chloride is all stablized, the aqueous solution of the gold chloride of 0.3ml is added drop-wise in the aqueous solution of HEPES.Reactant mixture was left standstill in 25 ℃ thermostat 1 hour.Make such collaurum nano particle thus, this collaurum nano particle comprises that the average grain diameter of projection is about 43nm, and is almost gold flat sugared shape, grafting shape or multiway shape (having 1-9 projection).The productive rate of colloidal solution per unit volume (0.1ml) is about 91%.Infer that residue comprises spherical particle or unreacted particle etc.

[growth phase]

To make and gold concentration is 4.0 * 10 by said method ^-4The colloid nuclear (5ml) of mol/L places the three-neck flask of 500ml, stirs to become 20 ℃ until fluid temperature in thermostat.Fluid temperature become stable after, drip the aqueous solution of gold chloride and the aqueous solution of 116ml L-ascorbic acid simultaneously with 1.0ml/ minute speed, wherein the aqueous solution of this gold chloride is by with 1.5 * 10 ^-2G(4.0 * 10 ^-5Mol) four hydration gold chlorides are dissolved in the ultra-pure water of 116ml and obtain, and the aqueous solution of this L-ascorbic acid is by with 4.2 * 10 ^-2G(2.4 * 10 ^-4Mol) L-ascorbic acid is dissolved in the ultra-pure water of 116ml and obtains.Make their reactions 2 hours by stirring, to carry out growth phase.After finishing dropping, three-neck flask taken out from thermostat and with its standing over night in freezer.The average grain diameter of the gold nano grain of Huo Deing (DLS) is about 66.5nm like this.Tem observation shows that the average core of this gold nano grain is of a size of a moon 25.7nm; Average projection length is 13.2nm; On average has the projection more than 4; The projection angle is about 50 degree; And AR is more than 1.The colloidal gold solution of Huo Deing is blue (based on the range estimation of Munsell colour system: form and aspect are approximately 5B) like this, and its maximum absorption wavelength is 610nm.

[embodiment 2]

Implement this embodiment with the synthetic flat sugared shape collaurum of gold with longer projection.

To become at the karyomorphism of embodiment 1 form in the stage and gold concentration be 4.0 * 10 ^-4The colloid nuclear (5ml) of mol/L places the three-neck flask of 500ml, stirs to become 20 ℃ until fluid temperature in thermostat.After fluid temperature is stable, drip the aqueous solution and the L-ascorbic acid of 116ml and the aqueous solution of HEPES of gold chloride simultaneously with 1.0ml/ minute speed, wherein the aqueous solution of this gold chloride is by with 1.5 * 10 ^-2G(4.0 * 10 ^-5Mol) four hydration gold chlorides are dissolved in the ultra-pure water of 116ml and obtain, and the L-ascorbic acid of this 116ml and the aqueous solution of HEPES are by with 4.2 * 10 ^-2G(2.4 * 10 ^-4Mol) L-ascorbic acid and 0.11g(4.0 * 10 ^-3Mol) HEPES is dissolved in the ultra-pure water of 116ml and obtains.Make its reaction 2 hours by stirring.Carry out growth phase thus.After finishing dropping, three-neck flask taken out from thermostat and with its standing over night in freezer.

The gold nano grain of Huo Deing comprises that the average grain diameter (DLS) of projection is about 98nm like this.Inferred by the tem observation result, formed the more substantial flat sugared shape collaurum of gold with longer projection.The average core of the flat sugared shape collaurum of the gold of these formation is of a size of about 65.7nm, and so the average length of the projection (grafting) of growth is about 16.7nm, has the projection more than 4, and the projection angle is about 50 degree, and AR is more than 1.The colloidal gold solution of Huo Deing is blue-green (based on the range estimation of Munsell colour system: form and aspect is approximately 8BG) like this, and its maximum absorption wavelength is 641nm.

[embodiment 3]

According to the synthetic collaurum of the mode identical with embodiment 1, difference is, changes the fluid temperature of growth phase into 10 ℃.The maximum absorption wavelength of thus obtained colloidal gold solution is shown in Table 1.

To become the 5ml colloid nuclear (4.3 * 10 that forms in the stage at the karyomorphism of embodiment 1 ^-4Mol/L) place the three-neck flask of 500ml, stir until reaching growth temperature in thermostat then, namely fluid temperature becomes 10 ℃.Drip the aqueous solution of gold chloride and the aqueous solution of 116ml L-ascorbic acid simultaneously with 1.0ml/ minute speed, wherein the aqueous solution of this gold chloride is by with 1.7 * 10 ^-2G(4.2 * 10 ^-5Mol) four hydration gold chlorides are dissolved in the ultra-pure water of 116ml and obtain, and the aqueous solution of this 116ml L-ascorbic acid is by with 4.2 * 10 ^-2G(2.4 * 10 ^-4Mol) L-ascorbic acid is dissolved in the ultra-pure water of 116ml and obtains.Make its reaction 2 hours by stirring.Carry out growth phase thus.After finishing dropping, three-neck flask taken out from thermostat and with its standing over night in freezer.

The gold nano grain of Huo Deing comprises that the average grain diameter (DLS) of projection is about 67nm like this.Tem observation shows that the average core of thus obtained gold nano grain is of a size of about 51.0nm, and the average length of the projection of growth (grafting) is about 8.0nm, has the projection more than 4, and the projection angle is about 50 degree, and AR is more than 1.The colloidal gold solution of Huo Deing is blue (based on the range estimation of Munsell colour system: form and aspect are approximately 5PB) like this, and its maximum absorption wavelength is 587nm.

[embodiment 4]

According to synthetic gold flat sugared shape, grafting shape or multiway shape (having 2-4 projection) the collaurum nano particle of being almost of the mode identical with embodiment 1, difference is, changes the fluid temperature of growth phase into 30 ℃.

The gold nano grain of Huo Deing comprises that the average grain diameter (DLS) of projection is about 60.5nm like this.Tem observation shows that the average length of the projection (grafting) of the growth of thus obtained gold nano grain is about 7.5nm, on average has the projection more than 4, and the projection angle is about 50 degree, and AR is more than 1.The maximum absorption wavelength of the colloidal gold solution of Huo Deing is 586.5nm like this.

The results are shown in the table 1.

[Comparative Examples 1]

According to the synthetic collaurum of the mode identical with embodiment 1, difference is, changes the fluid temperature of growth phase into 40 ℃.The maximum absorption wavelength of thus obtained colloidal gold solution is shown in Table 1.

Change the average grain diameter (DLS) that 40 ℃ of gold nano grains that obtain comprise projection into and be about 53nm by the temperature of growth phase is become.Tem observation shows that the average core of thus obtained gold nano grain is of a size of about 45nm, and the average length of the projection of growth (grafting) is about 4nm, on average has the projection more than 4, and the projection angle is about 10 degree.The colloid gold particle of Huo Deing is the polygonal (having 2-4 projection) with three-dimensional protrusion of little circle like this.Infer that residual fraction comprises spherical particle and unreacted particle.The colloidal gold solution of Huo Deing takes on a red color (based on the range estimation of Munsell colour system: form and aspect are approximately 10RP) like this, and its maximum absorption wavelength is 530nm.

[Comparative Examples 2]

According to the synthetic collaurum of the mode identical with embodiment 1, difference is, changes the amount of the ascorbic acid of growth phase into 2.1 * 10 ^-2G(1.2 * 10 ^-4Mol).The maximum absorption wavelength of thus obtained colloidal gold solution is shown in Table 1.

To become the 5ml colloid nuclear (4.3 * 10 that forms in the stage at the karyomorphism of embodiment 1 ^-4Mol/L) place the three-neck flask of 500ml, stir until reaching growth temperature in thermostat then, namely fluid temperature becomes 30 ℃.When fluid temperature is stablized, drip the aqueous solution of gold chloride and the aqueous solution of 116ml L-ascorbic acid simultaneously with 1.0ml/ minute speed, wherein the aqueous solution of this gold chloride is by with 1.7 * 10 ^-2G(4.2 * 10 ^-5Mol) four hydration gold chlorides are dissolved in the ultra-pure water of 116ml and obtain, and the aqueous solution of this 116ml L-ascorbic acid is by with 2.1 * 10 ^-2G(1.2 * 10 ^-4Mol) L-ascorbic acid is dissolved in the ultra-pure water of 116ml and obtains.Make its reaction 2 hours by stirring.Carry out growth phase thus.After finishing dropping, three-neck flask taken out from thermostat and with its standing over night in freezer.

The gold nano grain of Huo Deing comprises that the average grain diameter of projection is about 48nm like this.

The maximum absorption wavelength of the colloidal gold solution of Huo Deing is 536.3nm like this, and takes on a red color.

[Comparative Examples 3]

According to the synthetic collaurum of mode similar to Example 1, difference is, changes the amount of the ascorbic acid of growth phase into 8.4 * 10 ^-2G(4.8 * 10 ^-4Mol).The maximum absorption wavelength of thus obtained colloidal gold solution is shown in Table 1.

The average core diameter that changes 30 ℃ of gold nano grains that obtain by the temperature with growth phase into is 60.2nm, and average grain diameter is 70.2nm.The maximum absorption wavelength of the colloidal gold solution of Huo Deing is 550.0nm like this, and is orange.

[embodiment 5]

According to acquisition colloid gold particle similar to Example 2, difference is, uses HEPPSO to substitute HEPES, has obtained the colloid gold particle of average grain diameter for about 72nm.Thus obtained colloidal gold solution is rendered as blueness (based on the range estimation of Munsell colour system: form and aspect are approximately 1B), and its maximum absorption wavelength is 632nm.

[embodiment 6]

Prepare colloid gold particle according to mode similar to Example 2, difference is, uses PIPES to substitute HEPES, makes average grain diameter and is the colloid gold particle of about 81nm.Thus obtained colloidal gold solution is rendered as blueness (based on the range estimation of Munsell colour system: form and aspect are approximately 3B), and its maximum absorption wavelength is 626nm.

[embodiment 7]

According to the synthetic colloidal gold solution of mode similar to Example 2, difference is, the ascorbic acid that uses in the growth phase is replaced with 4.7 * 10 ^-2G(2.4 * 10 ^-4Mol) L-sodium ascorbate, and the consumption of HEPES is 0.22g(8.0 * 10 ^-3Mol).

The collaurum nano particle of Huo Deing comprises that the average grain diameter (DLS) of projection is about 82nm like this, average core is of a size of about 48nm, the average length of Sheng Chang projection (grafting) is about 20nm thus, on average has the projection more than 4, the projection angle is about 50 degree, and AR is more than 1.The colloidal gold solution of Huo Deing is navy blue (based on the range estimation of Munsell colour system: form and aspect is approximately 5PB) like this, and has the bigger maximum absorption wavelength of 752nm.

The measurement result of embodiment 1-7 and comparative example 1-3 all is shown in Table 1.

[table 1]

LAANa in the above form represents the L-ascorbic acid.

According to the synthetic colloidal gold solution of mode similar to Example 1, difference is, uses citric acid to replace ascorbic acid in growth phase.

Average grain diameter (DLS) and the average core size of the colloid gold particle that obtains among the average grain diameter that comprises projection (DLS) of the colloid gold particle of Huo Deing and average core size and each embodiment are in same level like this.The colloid gold particle that obtains among average length, average number and the angle of the projection of the growth of the colloid gold particle of Huo Deing (grafting) and each embodiment is in same level like this.The Ar of the colloid gold particle of Huo Deing is more than 1 like this.Therefore, the colloidal gold solution that obtains among the colloidal gold solution that obtains like this and each embodiment is in same level.This shows that other organic acids outside the organic acid that ascorbic acid or derivatives thereof or citric acid or derivatives thereof etc. can be had reproducibility are used as employed second reducing agent in the growth phase of the present invention.For example, according to inferring, the colloidal gold solution that obtains although use D (L)-malic acid, D (L)-tartaric acid, lactic acid, tannic acid or reduced sugar is different by a little with the colloidal gold solution that obtains according to the present invention, yet its character still is positioned within the preset range that satisfies purpose of the present invention.By using above-mentioned each sour inorganic salts or organic salt, according to described methods such as last embodiment 7, the colloidal gold solution that can obtain to be scheduled to.

Although example describes validity of the present invention by experiment below, yet the present invention is not limited to or is restricted to this.

＜carry out the experimental example that virus detects by immunochromatography 〉

[embodiment 8]

1. the preparation of the reacting part on the chromatography media

Use antibody coating machine (product of BioDot), phosphate buffer (pH7.4) dilution that will be contained 5 weight % isopropyl alcohols is coated nitrocellulose membrane (" HF120 " for the anti-A type influenza monoclonal antibody of the concentration of 1.0mg/ml, the product of Millipore) expansion direction upstream side (table 2:1 line), anti-Type B influenza monoclonal antibody is coated the downstream (table 2:2 line) of anti-A type influenza monoclonal antibody, then 50 ℃ dry 30 minutes down.After the drying, at room temperature dried overnight makes reacting part at the immunochromatography medium.

2. the preparation of mark substance solution 1

0.1ml is added in the blue collaurum suspension of the 0.5ml that obtains in embodiment 1 for the anti-Type B influenza monoclonal antibody of the concentration of 0.1mg/ml by phosphate buffer (pH7.4) dilution, the gained mixture was at room temperature left standstill 10 minutes.Then, add the phosphate buffer (pH7.4) that 0.1ml contains the bovine serum albumin of 10 weight %.After fully stirring, with 8000 * g with centrifugal 15 minutes of reactant mixture.Remove supernatant, add the phosphate buffer (pH7.4) that 0.1mL contains the bovine serum albumin of 1 weight % then, thereby obtain mark substance solution 1.

3. the preparation of mark substance solution 2

Add 0.1ml to 0.5ml collaurum suspension " the LC40 " (product of Tanaka's noble metal by phosphate buffer (pH7.4) dilution for the anti-A type influenza monoclonal antibody of the concentration of 0.1mg/ml, average grain diameter is 40nm) in, the gained mixture was at room temperature left standstill 10 minutes.Then, add the phosphate buffer (pH7.4) that 0.1ml contains the bovine serum albumin of 10 weight %.After fully stirring, with 8000 * g with centrifugal 15 minutes of reactant mixture.Remove supernatant, add the phosphate buffer (pH7.4) of the bovine serum albumin that contains 1 weight % then, thereby obtain mark substance solution 2.

4. the preparation of immunochromatography medium

The mark substance solution 1 and 2 of above preparation is evenly added on the mat of glass fibre system, dry in vacuum drier then, thus obtain to detect the reagent holding member.Then, with the chromatography media of preparation thus, detect the reagent holding member, will be as the sample pad of sample addition portion and the absorption pad that is used for absorbing expansion sample and insoluble carrier be laminated to the base material of backing plate system.At last, with cutting machine the gained duplexer is cut into the wide section of 5mm.

5. measure

Use the immunochromatography medium of above-mentioned preparation, according to following method antigen A) and Type B influenza virus (table 2: antigen B) analyze to whether there being A type influenza virus (table 2: in the sample.360000) and contain the developping solution that the tris buffer solution (pH8.0) of the sodium chloride of 1.0% bovine serum albumin and 150mM forms and be used as negative corpse or other object for laboratory examination and chemical testing sample be about to by 0.5% Tween20,0.6% polyvinyl pyrrolidone alkane (PVP) K-90(molecular weight:.Adding protein concentration in the gained developping solution is deactivation A type influenza virus and/or the Type B influenza virus of 25ng/mL, obtains positive corpse or other object for laboratory examination and chemical testing sample.Negative corpse or other object for laboratory examination and chemical testing sample and positive each 150mL of corpse or other object for laboratory examination and chemical testing sample are placed on the sample pad of immunochromatography medium and launch, carry out visual judgement after 15 minutes.At reactive site, the corpse or other object for laboratory examination and chemical testing sample of clearly observing luminous signal (luminescence signal) from detection line (Line 1 and No. 2 lines) is cited as "+"; When observing luminous signal, but its color is when very light, and this corpse or other object for laboratory examination and chemical testing sample is cited as " ± "; The corpse or other object for laboratory examination and chemical testing sample of not observing luminous signal is cited as "-".The result of embodiment 5 is as shown in table 2.

[table 2]

Be used in combination by the colloidal metal particle that routines such as the flat sugared shape colloid gold particle of gold of the present invention and spherical colloid gold grain are used, with the labelled reagent as immunologic assay, particularly immunochromatographic measurement, thereby make two kinds of different detection targets that contain in the organism sample clearly be detected the luminous signal for the detection line (Line 1 and No. 2 lines) that comes from reacting part respectively, this detection sensitivity is high and faulty identification can not occur.

Industrial applicibility

Colloid gold particle of the present invention is rendered as blueness, form agent or ammonium salt because it does not contain protective colloid, thereby do not have toxicity, and it contains the gold that is conducive to health.Therefore, can be with this colloid gold particle as pigment, cosmetics, immunologic assay labelled reagent, cytochemistry mark or protein staining agent.Particularly above-mentioned colloid gold particle is characterised in that:

(1) globose nucleus at colloid gold particle has 4-20 projection; And

(2) average grain diameter is 20nm to 200nm, can come mark and distinguish the detection target by visual blueness, therefore can be used as the immunologic assay labelled reagent in the immunochromatography inspection with two above color lines.

Although at length or with reference to some specific embodiment describe the present invention, yet it should be apparent to those skilled in the art that under the premise without departing from the spirit and scope of the present invention, can make various changes or modification.

The present invention is based on the Japanese patent application of submitting on November 5th, 2010 (Japanese patent application No.2010-248463), its content is incorporated this paper by reference into.The list of references of all references is incorporated this paper as a whole into.

Claims (according to the modification of the 19th of treaty)

1.(revise) a kind of blue gold nano grain, its average grain nuclear is of a size of 20nm to 60nm, average grain diameter is that projection and the projection length that 50nm to 120nm, each nuclear have more than four is 5nm to 50nm.

2. blue gold nano grain according to claim 1, wherein, the maximum wavelength of its ultraviolet-visible absorption spectroscopy is in the scope of 570nm to 800nm.

3. blue gold nano grain according to claim 1 and 2, wherein, described gold nano grain is the flat sugared shape particle of grafting shape particle, multiway shape particle or gold with three-dimensional protrusion.

4. according to any described blue gold nano grain in the claim 1 to 3, it obtains by the periphery growth that makes the nuclear that is made of gold nano grain.

5.(modification) a kind of colloidal solution of blue gold nano grain comprises: the blue gold nano grain described in the claim 1; The organic acid that contains piperazine ring for Good ' s buffer solution component; And the organic acid with reproducibility, and it is separated into colloidal solution.

6.(revise) a kind of method of making blue gold nano grain, comprising: karyomorphism becomes step, wherein, examines gold nano grain with the solution reaction of the first gold medal salt to form by making to the organic acid that contains piperazine ring of Good ' s buffer solution component; Growth step, wherein, the solution by in the solution of this nuclear gold nano grain, adding the second gold medal salt simultaneously and have the organic acid of reproducibility and make its reaction, thus this nuclear gold nano grain is grown.

7.(revise) method of the blue gold nano grain of manufacturing according to claim 6, wherein, described growth step be more than or equal to 10 ℃ and less than 40 ℃ reaction temperature under carry out.

8.(revise) according to the method for claim 6 or the blue gold nano grain of 7 described manufacturings, wherein, the described organic acid concentration in the described growth step is 0.075mM to 0.15mM.

9.(the modification) method of the blue gold nano grain of manufacturing according to claim 8, wherein, the described organic acid that contains piperazine ring for Good ' s buffer solution component is for being selected from by 2-[4-(2-ethoxy)-1-piperazinyl] ethyl sulfonic acid, 4-(2-ethoxy)-1-piperazine propane sulfonic acid, 4-(2-ethoxy) piperazine-1-(2-hydroxy propane-3-sulfonic acid), piperazine-1,4-two (2-ethanesulfonic acid), 3-[4-(2-ethoxy)-1-piperazinyl] propane sulfonic acid and piperazine-1, the organic acid of more than one of the group that 4-two (2-hydroxyl-3-N-morpholinopropanesulfonic acid) constitutes.

10.(the modification) method of the blue gold nano grain of manufacturing according to claim 6, wherein, described organic acid with reproducibility is more than one the organic acid that is selected from the group that is made of tartaric acid, tartrate, tannic acid, tannate, ascorbic acid, ascorbate, citric acid and citrate.

11.(revise) method of the blue gold nano grain of manufacturing according to claim 6, wherein, in described growth step, describedly be used in combination for the organic acid that contains piperazine ring and the described organic acid with reproducibility of Good ' s buffer solution component.

12.(revise) a kind of immunologic assay mark substance, it comprises any described blue gold nano grain in the claim 1 to 4.

13.(revise) mark substance for immunologic assay according to claim 12, it comprises at least two kinds of variform gold nano grains.

14.(revise) mark substance for immunologic assay according to claim 13, it comprises at least two kinds of variform gold nano grains, that is: spherical gold nano grain and grafting shape, multiway shape or the flat sugared shape gold nano grain of gold with three-dimensional protrusion.

15.(revise) a kind of immunologic assay method, it is used as mark substance with any described blue gold nano grain in the claim 1 to 4.

16.(deletion)

Claims

1. blue gold nano grain, it comprises that average grain diameter is the gold nano grain of 20nm to 200nm.

5. according to any described blue gold nano grain in the claim 1 to 4, its average grain nuclear is of a size of 20nm to 60nm, average grain diameter is that projection and the projection length that 50nm to 120nm, each nuclear have more than 4 is 5nm to 50nm.

6. the colloidal solution of a blue gold nano grain, it comprises: the blue gold nano grain described in the claim 1; The organic acid that contains piperazine ring for Good ' s buffer solution component; And the organic acid with reproducibility, and it is separated into colloidal solution.

7. method of making blue gold nano grain, comprising: karyomorphism becomes step, wherein, by making solution reaction for the organic acid that contains piperazine ring of Good ' s buffer solution component and the first gold medal salt to form the nuclear gold nano grain; Growth step, wherein, the solution by in the solution of this nuclear gold nano grain, adding the second gold medal salt simultaneously and have the organic acid of reproducibility and make its reaction, thus this nuclear gold nano grain is grown.

8. the method for the blue gold nano grain of manufacturing according to claim 7, wherein, described growth step be more than or equal to 10 ℃ and less than 40 ℃ reaction temperature under carry out.

9. according to the method for claim 7 or the blue gold nano grain of 8 described manufacturings, wherein, the described organic acid concentration in the described growth step is 0.075mM to 0.15mM.

10. the method for the blue gold nano grain of manufacturing according to claim 9, wherein, the described organic acid that contains piperazine ring for Good ' s buffer solution component is for being selected from by 2-[4-(2-ethoxy)-1-piperazinyl] ethyl sulfonic acid, 4-(2-ethoxy)-1-piperazine propane sulfonic acid, 4-(2-ethoxy) piperazine-1-(2-hydroxy propane-3-sulfonic acid), piperazine-1,4-two (2-ethanesulfonic acid), 3-[4-(2-ethoxy)-1-piperazinyl] propane sulfonic acid and piperazine-1, the organic acid of more than one of the group that 4-two (2-hydroxyl-3-N-morpholinopropanesulfonic acid) constitutes.

11. the method for the blue gold nano grain of manufacturing according to claim 7, wherein, described organic acid with reproducibility is more than one the organic acid that is selected from the group that is made of tartaric acid, tartrate, tannic acid, tannate, ascorbic acid, ascorbate, citric acid and citrate.

12. the method for the blue gold nano grain of manufacturing according to claim 7 wherein, in described growth step, describedly is used in combination for the organic acid that contains piperazine ring and the described organic acid with reproducibility of Good ' s buffer solution component.

13. an immunologic assay mark substance, it comprises any described blue gold nano grain in the claim 1 to 5.

14. the mark substance for immunologic assay according to claim 13, it comprises at least two kinds of variform gold nano grains.

15. the mark substance for immunologic assay according to claim 14, it comprises at least two kinds of variform gold nano grains, that is: spherical gold nano grain and grafting shape, multiway shape or the flat sugared shape gold nano grain of gold with three-dimensional protrusion.

16. an immunologic assay method, it is used as mark substance with any described blue gold nano grain in the claim 1 to 5.